The long term goals of this project are to understand the structure and function of physiologically and clinically important human facilitated glucose transporters (Gluts), members of the Major Facilitator Superfamily (MFS), through a combination of X-ray crystallography and biochemical/biophysical approaches. Glut1, which is abundant in the human red blood cells (RBCs) and the blood-brain barrier, is an extensively-studied representative of these facilitated transporters. Glut4 is responsible for insulin-regulated glucose disposal. Several diseases have been identified with mutations resulting in malfunction of Gluts, such as GLUT1 deficiency syndrome and type II diabetes. It is important to obtain a high-resolution structure, as well as characterize the transport mechanism at an atomic level. Thus far, there is no 3-D crystal structure available for any glucose facilitator, although many attempts have been made, particularly with Glut1. Protein production and crystallization are two major bottlenecks for the structural determination. The PI of this application has demonstrated that manipulating phospholipids (PL) improves the quality of crystals of the lactose permease from Escherichia coli, a paradigm for the MFS, and several other hydrophobic membrane proteins. To test the hypothesis that PL and/or neutral lipids play an important role in crystallization of human Glut facilitators, the PI has optimized a simple method to obtain Glut1 by differential isolation from RBC ghost membranes, which are prepared from out-dated human RBCs. Furthermore, a dedicated system to obtain the overexpression of human Glut1 and Glut4 in Sachromyces cerevisiae has also been achieved. Specific aims of this application include: 1) Optimization of protein production by both recombinant expression and differential isolation to increase quantity and purity. Different affinity tags at different locations with each Glut will be screened for expression, purification, stability and function. 2) Characterization of PL and neutral lipids with respect to crystal quality. The important role(s) of PL and/or neutral lipids for function, stability, as well as crystal quality will be systematically tested. Attention will be focus on the native lipids. 3) Optimization of a crystallization procedure for X-ray structure determination. Crystal structure of any Gluts will significantly contribute to our understanding of transport mechanism and lay the foundation for rational new drug design and therapeutic intervention. Furthermore, the structure is also expected to provide valuable information in defining the role of lipids in crystallization of membrane proteins. Relevance Statement: Successful crystallization of eukaryotic Gluts is imperative to obtaining X-ray crystal structures;the expected structures will substantially improve our understanding of facilitated glucose transport and provide important clues for therapeutic intervention, which will have significant impact in biology and medicine. Characterization of role(s) of PL or neutral lipids in crystallization of Gluts will help in the establishing of a basic guideline for application of the novel approach in other membrane protein crystallization.